Molecular breeding technology for crops makes it possible to use the genes of all species as breeding material and to regulate the effects of breeding minutely at the gene level instead of at the genome level as in the past. Therefore it is one of the core technologies leading into the next generation of agriculture.
In order to maximize the effects of such molecular breeding technologies for crops, the essential prerequisites are as follows:
1) The accumulation of a database of genes to represent various plants;
2) The establishment of transformation systems for various crops; and
3) The development of promoters that regulate the expression of foreign genes inserted into plants.
In foreign countries promoters regulating the expression of plant genes have been studied since the early 1980's. It has been suggested that a promoter of cauliflower mosaic virus could induce high levels of gene expression in all kinds of plant tissues (Hohn et al., 1982, Cwrr. Topics Microbiol. Immunol. 96: 193-236).
Subsequently, the sequence of the promoter was identified (Odell et al., 1985, Nature 313:810-812). It was proven that the promoter could induce high levels of gene expression in plants (Sanders et al., 1987, Nucleic Acids Res. 15: 1543-58). Since then, CaMV 35S promoter (Patent NO.: JP1993192172-A1) has become the most universal promoter used in plants.
Since the identification of CaMV 35S, other inducible promoters whose activities increase under biotic or abiotic conditions have been actively studied.
Especially, sucrose-inducible promoters whose activities increase upon treatment with sucrose have been actively studied. Such sucrose-inducible promoters have merits when applied to the mass production of useful medicinal and industrial proteins in plant storage organ tissues, such as storage roots, which contain starch synthesized from sucrose in relatively large quantities. Such useful proteins include very valuable and expensive medicinal or industrial proteins, such as interferon, growth hormones, Lactoferrin, and phytase.
The studies on sucrose-inducible promoters have been focused on genes coding storage proteins accumulated in storage organ tissues or genes relating to the synthesis of starch.
For example, patatin is a storage protein in the potato. It has been identified that the activity of a patatin gene promoter is increased by sucrose (Rocha-Sosa et al., 1989, EMBO J 8, 23-31; Wenzler et al., 1989a, Plant MoI. Biol. 12, 41-50; Wenzler et al., 1989b, Plant MoI. Biol. 13, 347-354). It has also been reported that a specific nucleotide sequence of the −344 region of the promoter (B sequence) plays an essential role in sucrose induction (Grierson et al., 1994, Plant J, 5, 815-826).
Meanwhile, there are two sucrose synthase genes, Sus3 and Sus4, in the potato. It has been identified that Sus4, among the two genes, is expressed by sucrose (Salanoubat and Belliard, 1989, GENE 84, 181-185). In addition, it has been reported that the −1500˜−267 region of the Sus4 gene promoter, the 3′ untranslated region, and a 1612 bp leader intron are essential for sucrose induction (Fu et al., 1995, Plant Cell 7, 1387-1394).
Furthermore, it has been identified that the activity of a starch-branching enzyme I (SBE1) gene promoter in corn is increased by sucrose, and that a sequence between −314 and −145, relative to the transcription initiation site of the gene, is essential for sucrose induction (Kim and Guiltinan, 1999, Plant Physiology 121, 225-236).
In addition, it has been reported that the activity of a β-amylase gene promoter in sweetpotato is inducible by sucrose and that a sequence between −901 and −820, relative to the transcription initiation site of the gene, is essential for sucrose induction. Furthermore, a TGGACGG sequence therein plays an important role as a regulator (Maeo et al., 2001, Plant MoI. Biol. 46, 627-637).
Meanwhile, ADP-glucose pyrophosphorlyase is believed to be the key regulatory enzyme in controlling the amount of starch in plants. The ADP-glucose pyrophosphorlyase gene promoters in tomato and Arabidopsis have been reported to be sucrose-inducible (Siedlecka et al., 2003, Planta 217, 184-192; Li et al., 2002, Plant Science 162, 239-244). In addition, it has been reported that the activity of the promoter in Arabidopsis is decreased by okadaic acid, which is an inhibitor of protein phosphatase I and 2A.
Meanwhile, there are two ADP-glucose pyrophosphorlyase genes, ibAGP1 and ibAGP2, in sweetpotato. It has been reported that the expression of the ibAGP1 gene (the gene was previously named ibAGP-sTL1) is enhanced by sucrose (Bae and Liu, 1997, Molecular Genetics and Genomics 154, 179-185). The full nucleotide sequences of the above two genes have been analyzed and compared, and the transcription initiation site of each of the genes has been identified (Noh et al., in press, GENE).
However, nucleotide sequence of highly efficient sucrose-inducible promoter has not been reported. Therefore, there is an increasing need for sucrose-inducible promoters that can play important roles in the cheap and efficient production of useful foreign proteins in large quantities in plants.